Project A. Determine genetic susceptibility and immunopathological mechanisms contributing to idiopathic tooth root resorption Background: Previously, we reported that BSP KO mice exhibit a tooth root resorption phenotype. Based on this finding, we sought to identify human subjects exhibiting multiple idiopathic cervical root resorption (MICRR), a familial pattern of MICRR with suggested genetic susceptibility. Following IRB approval from the University of Detroit Mercy School of Dentistry and NIH, dental/medical histories, x-rays, saliva samples, and extracted teeth were collected from a kindred (4 affected and 4 unaffected members) exhibiting MICRR. On examination, the proband and the affected son and daughter exhibited severe root resorption of multiple teeth, with no other significant medical history. Micro-CT of exfoliated teeth revealed severe cervical root resorption distinct from tooth decay. Whole exome sequencing using saliva from affected and unaffected family members identified SNPs in ten candidate genes that co-segregated with the resorption phenotype, including a novel autosomal dominant missense mutation in the Interferon Regulatory Factor 8 (IRF8) gene. Detailed analysis identified a mutation in the c-terminal region of IRF8 responsible for overactive osteoclast function. Results were published in JBMR, 2019 (see below). This project transitioned to University Maryland with Dr. Thumbigere-Math (K99/R00 recipient). Ongoing: We continue to collaborate with Dr. Thumbigere-Math as the lead on the IRF8 project, with other NIH IRF8 collaborators, Drs. Ozato and Holland. In addition, we have established a collaboration with a periodontist in Chicago, IL, Dr. Steve Russo related to a patient of his that developed idiopathic root resorption within a two-year period (2017-2019). We will be analyzing teeth, tissues, saliva and plaque samples. We have set up a collaboration with an expert in microbial analyses, Dr. Purnima Kumar at the Ohio State University, to determine the microbial profile of plaque samples from this patient. Further, we are working with Dr. Betty Hajishengallis at U. Penn related to a patient with missing ossicles and idiopathic root resorption. The patient, aged 10 yrs., recently had genetic testing and was diagnosed with familial expansile osteolysis. We are working with Dr. Hajishengallis to determine the mechanism for root resorption, including a potential link to alternations in specific genes noted in this patient. Project B. Disorders of mineralization: In collaboration with NIDCR clinical researchers and other IC clinicians, we have been examining individuals with mineralized tissue metabolism disorders for alterations in tissues/cells of the DOC complex. 1.Mutations in key regulators of Pi/PPi. Mineralization of skeleton and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. We examined the effect of decreased PPi on development and maturation of teeth in human subjects (4) with generalized arterial calcification of infancy (GACI), who harbor loss-of-function mutations in the ENPP1 gene. Three of the four subjects reported a history of infraocclusion or over-retained primary teeth or poor orthodontic tooth movement, suggesting altered mineral metabolism as a contributing factor. All subjects presented radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. Micro-CT analyses of extracted primary teeth from two GACI subjects revealed marked increase cervical cementum thickness and density vs. age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 knock-out mouse molars revealed a marked increase in acellular cementum thickness and volume. Collectively, these findings report a novel dental phenotype in GACI and further support our hypothesis that Pi/PPi modulation is as a key mechanism for regulating cementogenesis across species. Thumbigere-Math V et al., JDR, 2018. Ongoing: We have continued to examine patients with Pi/PPi disorders at NIH CRC as well as analyzing exfoliated or extracted teeth from patients with Pi/PPi disorders. We have also included patients with ABCC6 (ATP Binding Cassette Subfamily C Member 6), which is associated with some cases of GACI. For controls, in collaboration with North Carolina State University and Duke under the Newborn Epigenetics Study, we are obtaining exfoliated primary teeth from healthy individuals.